KR20170028044A - Novel Bacillus amyloliquefaciens CJ 14-6 isolated from the Korean traditional meju and manufacturing method of soybean Koji using this fungi and soybean Koji manufactured therefrom - Google Patents

Abstract

The present invention relates to a Bacillus amyloliquefaciens CJ 14-6 strain, which is isolated from Korean traditional soybean lumps, a method for manufacturing soybean koji using the same and soybean koji manufactured thereby. The method for manufacturing soybean koji comprises: a steaming step of immersing soybean or adding water in soybean, and steaming soybean; and a fermenting step of inoculating a Bacillus amyloliquefaciens CJ 14-6 strain to the steamed soybean, fermenting and drying the same, and making soybean koji.

Description

[0001] The present invention relates to a novel strain isolated from a traditional meju, a soybean curd prepared by using the same, and a soybean curd prepared by the method, wherein the soybean curd {Novel Bacillus amyloliquefaciens CJ 14-6 is isolated from the Korean traditional meju and manufacturing method of soybean koji using this fungi and soybean Koji manufactured therefrom}

The present invention relates to a novel strain, Bacillus amyloliquefaciens CJ 14-6, which has excellent protease activity and has anti-obesity activity, isolated from a traditional meju, and soybean Koji, And a soybean curd produced by the method.

Meju is a fermented food used as a raw material for traditional soybean fermented foods such as soy sauce, kochujang and miso. Meju, which is mainly made from soybeans, is also an important starter for the production of conventional soy sauce, such as soy sauce, red pepper paste and miso.

Meju is classified into conventional meju, improved meju and industrial co-op according to the manufacturing method. Conventional meju is formed by using only soybean, then wrapped in straw and fermented for a certain period of time. The modified meju is fermented with a strain of Aspergillus sp. The wheat flour was fermented by Hwangguk-gyun in Aspergillus.

Kochujang is largely divided into traditional (traditional) kochujang and factory (improved) kochujang depending on the manufacturing method. Traditional kochujang is made by mixing fermented soybean meal with soybean paste and red pepper powder, starch raw materials such as meju and rice for kochujang made by mixing soybean and cereal at a certain ratio. The factory-made kochujang is a maturing formula, a saccharified kochujang, and a koji which is a pure culture of Aspergillus oryzae instead of a meju for kochujang. Soy sauce is used as a raw material for protein, and rice or wheat is used as a raw material for starch.

As the number of women entering the society increases, the number of households purchasing the kochujang produced at the factory is increasing, and the production of such koshuchang is continuously increasing, and the export amount of kochujang is also continuously increasing.

Korean Patent No. 10-0668056 discloses a soybean meju prepared by inoculating microorganisms pre-cultured on a bean bean and fermenting the bean bean as a method for mass production of the conventional plant-based kochujang, and a production method thereof. More specifically, the prior art includes a step of growing soybeans and drying them, and a fermentation step of fermenting the soybeans for a certain period of time after inoculating the microorganisms cultured separately from the traditional meju. And a soybean meju produced by the method.

However, even with the above-mentioned conventional methods, there is still a demand for the development of bean curd for mass production of factory-style kochujang.

KR 10-0668056 B1 (Public Notice 2007.01.11)

Accordingly, the present inventors selected strains of Bacillus amyloliquefaciens CJ 14-6 by screening strains having excellent protease activity in a conventional soybean paste as a study for mass production of a plant-based kochujang, It is possible to produce a soybean curd suitable for mass production of a plant-based kochujang, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a strain of Bacillus amyloliquefaciens CJ 14-6 isolated from a traditional meju.

Another object of the present invention is to provide a method for producing soybean curd using the strain.

It is still another object of the present invention to provide a soybean curd produced by the above production method.

In order to achieve the object of the present invention, the present invention provides a strain of Bacillus amyloliquefaciens CJ 14-6, which is a strain having excellent protease activity and anti-obesity activity isolated from a traditional meju .

The present invention also relates to a method for producing soybean milk, comprising the steps of: And an empowering step of inoculating and fermenting the Bacillus amyloliquefaciens CJ 14-6 strain to the expanded soybeans.

The present invention also provides a bean curd made by the method of manufacturing the bean curd.

The present invention relates to a method for producing a large amount of a plant-type kochujang by separating and selecting a novel strain Bacillus amyloliquefaciens CJ 14-6 having excellent protease activity from a traditional meju and using the same in the production of soybean curd .

Hereinafter, the present invention will be described in detail.

As a first aspect of the present invention, the present invention provides a strain of Bacillus amyloliquefaciens CJ 14-6, which is a strain having excellent protease activity isolated from a traditional meju.

Specifically, the present invention relates to a method for isolating various strains from Korean traditional meju, followed by first screening of a strain of Bacillus spp., Which has excellent protease activity, from an active medium, and secondly, And the strains having strong proteolytic ability were secondly selected. 16s rDNA sequencing (16s rDNA sequencing) was performed to identify the final selected Bacillus spp.

The isolated strain of Bacillus was identified as Bacillus amyloliquefaciens (SEQ ID NO: 1). This strain having excellent protease activity was named Bacillus amyloliquefaciens CJ 14-6 and deposited on July 1, 2015 with the Korean Society for Microbiological Protection (Accession No. KCCM11718P).

According to a second aspect of the present invention, there is provided a method for producing soybean milk, comprising the steps of: And an empowering step of inoculating and fermenting the Bacillus amyloliquefaciens CJ 14-6 strain to the expanded soybeans.

More specifically, the present invention relates to a method for producing soybean milk, comprising the steps of selecting and washing soybean, dipping soybeans or adding soybean to soybeans; Growing the soybeans and cooling them; Culturing Bacillus amyloliquefaciens CJ 14-6 to prepare a culture; And a step of inoculating the cooled soybeans with a culture solution of the strain of Bacillus amyloliquefaciens CJ 14-6, and a step of emulsifying the fermented soybeans.

Further comprising the step of immersing the soybeans selected and washed before the soybean-growing step at a temperature of 10 to 50 ° C for 1 to 15 hours, and adding saturated steam (1.0 to 2.0 kgf / cm ² ) 150 ° C for 1 to 60 minutes, but is not limited thereto. The expanded soybeans can be cooled to about 30 캜 to 50 캜, more specifically about 30 to 35 캜.

The beans can be grown in a high pressure steam sterilizer (autoclave) at 100 ° C to 150 ° C for 5 to 15 minutes, more specifically at 110 ° C for 10 minutes.

The Bacillus amyloliquefaciens CJ 14-6 strain can be used as a culture solution which is cultured by being transformed into a spore state. The culture solution can be uniformly inoculated into the expanded soy beans at 0.1 to 3.0 wt% based on the total amount of the raw materials.

The culture medium for culturing the strain may be a soy sauce medium. Wherein the soy sauce medium comprises 1 to 10% of liver selected from Korean soy sauce, brewed soy sauce or mixed soy sauce and 0.1 to 10% of a sugar source selected from the group consisting of glucose, sucrose, glactose or maltose, . ≪ / RTI >

In another embodiment of the present invention, the present invention relates to a method for producing Bacillus amyloliquefaciens CJ 14-6 strain inoculated in a soy sauce medium (brewed soy sauce + glucose) Was cultured for 20 to 42 hours to prepare a culture broth of Bacillus amyloliquefaciens CJ 14-6.

Bacillus amyloliquefaciens CJ 14-6 strain is inoculated and mixed, and then fermented at 30 ° C to 45 ° C, more specifically 34 ° C to 44 ° C, for 1 to 3 days to produce soybean curd.

In addition, the fermentation step may be followed by a drying step.

As a third aspect of the present invention, the present invention also provides a bean curd made by the method for manufacturing the bean curd.

The soybean curd produced by the production method of the present invention can be used for the mass production of the plant-made kochujang.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[Example]

Example 1: Isolation and identification of strains from traditional meju

(1) Isolation and Identification of Strain

The strains used as starters in the present invention were isolated and sorted from traditional meju collected from traditional food manufacturers in Gyeonggi, Gangwon, Chungbuk, and Chonnam.

Traditional Meju was diluted in sterilized water and then plated on agar medium (Nutrient agar, Difco) and cultured at 37 ℃. Five bacterium species were selected from traditional meju and identified by pure separation. The isolated strains were named CJ 3-27, CJ 4-4, CJ 5-10, CJ 14-6 and CJ 16-57, respectively.

The isolated strains were cultured in nutrient broth (Nutrient Broth, Difco) at 37 ° C for 24 hours at 200 rpm, and then the titers of proteases were compared. The results of the identification and the potency of the protease are shown in Table 1 below.

 Identification of isolates derived from traditional meju Primary screening strain Protease activity (U / g) Identification result CJ 3-27 62.58 + 0.17 Bacillus amyloliquefaciens CJ 4-4 91.44 + - 6.97 Bacilus licheniformis CJ 5-10 61.72 + - 4.13 Bacillus subtilis subsp . Subtilis CJ 14-6 140.73 + - 4.62 Bacillus amyloliquefaciens CJ 16-57 222.42 + 0.63 Bacilus licheniformis

(2) Primary selection

The isolates CJ 3-27, CJ 4-4, CJ 14-6, and CJ 16-57 were selected as the first strains except CJ 5-10, which exhibited the lowest protease activity among the identified strains.

(3) Secondary selection

Soybean curds were prepared using the four strains selected first and the protease activity of each strain was measured. The results are shown in Table 2 below, and the excellent strains were secondly selected by comparing them.

For the production of soybean curd, 1 kg of soybean was immersed in purified water for 12 hours, and then it was heated at 110 ° C for 10 minutes by autoclave, and after heating, it was cooled to 35 ° C. The separated strains were mixed in the amount of 2.0 wt% based on the total amount of the raw materials, and then cultured at 37 for 3 days, respectively, to prepare soybean curds.

Protease activity was measured by extracting each of the soybean curd prepared above with distilled water at 30 ° C for 1 hour and filtering it as a crude enzyme solution. 0.5 ml of crude enzyme solution, 1.5 ml of 2% milk casein and 1 ml of Mcllivine buffer (pH 6.0) were added to the enzyme reaction solution, followed by reaction at 38 ° C for 1 hour. Thereafter, 3 ml of 0.4 M TCA solution was added to stop the reaction. After filtration, 5 ml of 0.4 M Na ₂ CO ₃ and 1 ml of a phenol reagent were added and mixed thoroughly. The mixture was developed at 38 ° C for 30 minutes, Absorbance was measured at 660 nm. At this time, the enzyme activity was defined as one unit of the amount of enzyme producing tyrosine corresponding to 1 占 퐂 per minute, and a calibration curve was prepared using tyrosine as a standard substance.

Comparison of Protease Activity of Soybean Cultivars Applied to Selected Primary Strain First selected strain Protease activity (U / g) CJ 3-27 258 CJ 4-4 118 CJ 14-6 225 CJ 16-57 155

From the results shown in Table 2, CJ 3-27 and CJ 14-6 strains having excellent protease activity were selected.

(4) tertiary selection - in vitro 3T3-L1 anti-obesity activity

A. Cell line

3T3-L1 cells, a mouse embryonic fibroblast cell line, were distributed from Korean Cell Line Bank (KCLB).

B. Cell culture

3T3-L1 cells were cultured in DMEM medium containing 10% BCS, 1% penicillin-streptomycin. Cells were recovered by centrifugation at 1,000 rpm for 5 minutes when cultured at 70% level in a culture dish.

MTT  assay

The MTT assay was performed according to the method of Carmichael et al. Based on that mitochondria dehydrogenase in living cells reacted with MTT to form a dark blue MTT formazan crystal. 3T3-L1 cells were seeded at a concentration of 1.5 x 10 ⁴ / ml in 500 wells in 48 wells and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours so that the cells could be attached to the plate bottom. The next day, samples were injected into each well to a final concentration of 1000, 250, and 0 μg / ml. After culturing for 24 hours, the medium was adjusted to 5 mg / ml in the medium containing the sample, and 50 μl of thiazolyl blue tetrazolium bromide solution was added thereto. The final concentration was adjusted to 500 μg / ml, % CO ₂ incubator for 4 hours. After the reaction was completed, the medium containing the MTT reagent was removed, and 300 μl of DMSO (dimethyl sulfoxide) was added to dissolve the MTT-formazan crystal, followed by induction of color development for 5 minutes. Then, using an ELISA microplate reader, The absorbance was measured.

LA3T3 -L1 induction of cell differentiation

To induce cell differentiation, cells were inoculated at 2.5 x 10 ⁴ / ml in 6-well plates and cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin until a post-confluent state of 4 And cultured in a 5% CO ₂ incubator at 37 ° C for a day. Representative positive controls used in anti-obesity experiments include resveratol, which inhibits the differentiation of whole adipocytes, stimulates lipolysis, induces apoptosis of mature adipocytes and inhibits lipogenesis.

In the post-confluent state, Day 0 was supplemented with 10% FBS, 1% penicillin-streptomycin, 5 μg / ml insulin, 1 μM DMS (dexamethasone), 0.5 mM IBMX (3-isobutyl- 1methylxanthine was treated for 72 hours and Day 3 and 5 were treated with DMEM differentiation medium containing 10% FBS, 1% penicillin-streptomycin and 10 μg / ml INS. At the same time, every time the medium was changed, all samples were treated to a final concentration of 10, 50, 0 μg / ml and treated with a positive control, 20 μg / ml.

E.Oil  Red O dyeing and quantification

To determine the degree of differentiation of cultured 3T3-L1 cells, Oil Red O reagent, which stains fat droplets on the 7th day of the differentiation, was modified and stained with Rene and Kasturi.

 The differentiated cells were fixed with 4% para-formaldehyde (in PBS) at room temperature for 1 hour or more. Cells were stained with 0.5% Oil Red O staining for 1 hour and washed twice in running water to remove remaining staining to ensure that the staining reagent did not remain on the plate. Dyed droplets were dissolved in isopropanol Absorbance was measured at 510 nm.

In vitro anti - obesity activity was measured by comparing the inhibitory effect of two kinds of Bacillus amyloliquefaciens CJ 3-27 and CJ 14-6, which were confirmed to have high protease activity, on adipocyte differentiation.

Inhibition rate of adipocyte differentiation of secondary selected strains Strain Concentration of culture medium
(Μ g / ml) Rate of differentiation inhibition
(%) Bacillus amyloliquefaciens
CJ 3-27 25 10.10 50 6.98 100 3.37 Bacillus amyloliquefaciens
CJ 14-6 25 20.57 ^* 50 26.41 ^* 100 36.61 ^*

Statistical tests: Student-T test

*: P < 0.05

As shown in the above Table 3, inhibition rate of adipocyte differentiation of CJ14-6 culture medium was significantly inhibited compared to that of CJ 3-27 culture medium in which the inhibition rate of adipocyte differentiation was not significantly changed.

Example  2 : Bean  Produce

A soybean curd was prepared according to the soybean curd production method described in Example 1- (1) above using the newly selected strain, Bacillus amyloliquefaciens CJ 14-6, finally selected in Example 1 as a seed strain . Respectively.

For comparison with soybean curd using conventional Aspergillus oryzae , in Example 1- (1), Aspergillus oryzae commercially available from Chungmu fermentation was used as Aspergillus , The soybean curd was prepared according to the soybean curd production method described in &quot;

(1) Protease activity measurement

The protease activity of each soybean curd was measured and the results are shown in Table 4 below.

The enzyme titer of the new Bacillus amyloliquefaciens CJ 14-6 and the conventional Aspergillus oryzae bean curd Strain Protease activity (U / g) Bacillus Amylori Qua Faces CJ 14-6 225 Conventional Aspergillus oryzae 102

From the results in Table 4, it was found that the protease enzyme activity of the bean curd using the novel strain of Bacillus amyloliquefaciens CJ 14-6 was 120.6% higher than that of the bean curd using Aspergillus oryzae , Can improve the decomposition ability of the protein component when used in the koji paste or doenjang to improve the taste quality.

Experimental Example 1: Application of new strain (CJ14-6)

Animal experiments were conducted to verify the anti-obesity efficacy of the new Bacillus amyloliquefaciens CJ 14-6 soybean curd (Example 4). The animals used in the experiment were purchased from Darussian (Korea) with male rats (5-week-old SD rats), maintained at 18 ± 2 ℃ and illuminated 12 hours (08: 00 ~ 20: 00) Respectively.

Experimental rats were divided into two groups of 7 rats. In the control group, 20% of lard (lard) was added to the rat powder feed, and the high fat diet was fed. In the high fat diet group, Application Soybean curd powder was fed 0.91%. Weight and fat tissue weights for these are shown in Tables 5 and 6.

Body weight and dietary intake were measured every week, and fat tissue weight and lipid content were measured after fasting for 12 hours before the end of the test. The collected blood was centrifuged at 1,900 × G for 20 minutes, and the serum was separated and used as a serum lipid content sample. To analyze the total lipid, triglyceride and total cholesterol content of hepatic and adipose tissues, 0.1 g of liver and adipose tissues were added with chloroform-methanol (2: 1, v / v) After left standing, distilled water was added and the total lipid content in the lower layer of the lipid layer was measured after centrifugation at 1,150 x G for 20 minutes. This total lipid was diluted and used for the analysis of total cholesterol and triglyceride content. The results of lipid content of serum and tissues are shown in Tables 7, 8, 9 and 10.

To measure the fatty acid biosynthesis-related enzymatic activity of adipose tissue, 0.1M potassium phosphate buffer (pH 7.4, 37 ° C) corresponding to 3 times the weight of adipose tissue was added and homogenized. The resulting solution was incubated at 3,000 rpm for 15 minutes After centrifugation, the supernatant was again centrifuged at 15,000 rpm for 30 minutes, and then the supernatant was collected. The results of enzyme activity are shown in Tables 11 and 12.

For the measurement of adipocyte size, paraffin blocks were prepared by fixing the adipose tissue with a Bouin solution, and slides were prepared and stained with anillin blue staining solution. After the photograph of the adipocyte was taken through the electron microscope, the adipocytes in the treatment zone were compared.

Treated group Starting weight (g) Finished weight (g) Weight gain (g / day) Dietary intake (g / day) Control group 171.79 544.00 6.84 0.40 17.49 + - 0.80 Example 2 Group 172.57 513.29 6.26 ± 0.40 17.33 ± 1.78

Treated group Fat tissue weight (g / 100g body wt.) Epididymis Fat around the kidney Total white fat Control group 2.43 0.78 6.80 Example 2 Group 2.24 0.65 6.29

Treated group Lipid content in serum (mg / dl) Triglyceride Total cholesterol LDL-cholesterol Control group 225.57 ± 25.64 128.43 + - 12.92 125.40 ± 15.43 Example 2 Group 152.57 ± 10.21 ^*** 95.86 +/- 8.28 ^*** 63.66 ± 8.75 ^***

Treated group Lipid content in liver tissue (mg / g) Total fat Triglyceride Total cholesterol Control group 247.02 + - 21.87 94.91 + - 18.43 360.45 ± 57.55 Example 2 Group 220.99 ± 16.77 ^* 64.57 ± 3.00 ^** 292.48 + - 76.68

Treated group Lipid content in epididymal adipose tissue (mg / g) Total fat Triglyceride Total cholesterol Control group 501.55 ± 57.14 166.15 + 16.18 294.90 ± 15.00 Example 2 Group 459.50 ± 35.17 118.44 ± 18.44 ^*** 250.48 ± 26.74 ^**

Treated group Lipid content in mesenteric adipose tissue (mg / g) Total fat Triglyceride Total cholesterol Control group 412.22 ± 37.59 122.01 + - 6.02 178.23 ± 24.41 Example 2 Group 368.37 ± 23.66 ^* 108.81 ± 9.02 ^** 147.77 ± 12.72 ^*

Treated group Enzymes related to fatty acid synthesis in liver tissues (nmol / min / mg protein) FAS ME G6PDH Control group 12.22 + - 1.07 31.47 ± 1.39 24.68 ± 1.45 Example 2 Group 9.77 + - 0.54 26.96 ± 1.74 17.31 ± 1.19 ^**

Treated group Enzymes related to fatty acid synthesis in fatty tissue (nmol / min / mg protein) FAS ME G6PDH Control group 7.29 ± 0.59 8.00 0.31 24.62 ± 1.26 Example 2 Group 5.35 0.35 ^* 5.51 + - 0.62 ^** 20.21 + - 1.30 ^*

Treated group Control group Example 2 Group Fat cell photograph

Fat Cell Size (μm ² ) 21.41 ± 2.45 18.18 ± 1.11

Treated group Measurement of body fat accumulation index factor Leptin (μg / ml) HR-LPL (Unit / g) TE-LPL (Unit / g) Control group 5.75 + - 0.70 7.03 + - 1.08 36.12 + - 5.50 Example 2 Group 3.97 ± 0.17 ^*** 4.30 ± 1.06 ^*** 22.74 ± 2.08 ^***

Statistical tests: Student-T test

*: P < 0.05

**: P < 0.01

***: P < 0.001

As shown in the results of Table 5, it was confirmed that the weight gain of the group of Example 2 was 91.5% higher than that of the control group. In addition, as shown in Table 6, the weight gain of peripheral fat tissue was reduced to 83.3% as compared with the control.

Therefore, it can be seen from the results of the above example that the soybean strain of the new strain (CJ14-6) has a weight loss effect.

Korea Microorganism Conservation Center (overseas) KCCM11718P 20150701

<110> CJ Cheiljedang Corporation <120> Novel Bacillus amyloliquefaciense CJ 14-6 isolated from the          Korean traditional meju and manufacturing method of soybean Koji          using this fungi and soybean Koji prepared therefrom <130> PA14-0407 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1418 <212> DNA <213> Bacillus amyloliquefaciens <400> 1 agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac 60 gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgg 120 ttgtttgaac cgcatggttc agacataaaa ggtggcttcg gctaccactt acagatggac 180 ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcgacgat gcgtagccga 240 cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300 gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360 aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtgccgtt caaatagggc 420 ggcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta 480 atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc 540 ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact 600 tgagtgcaga agaggagagt gggagtacca cgtgtagcgg tgacatgcgt agagatgtgg 660 aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg agcgacagcg 720 tggggagcga acaggattag ataccctggt agtccacgcc gtaaacgatg agtgataagt 780 gttagggggt ttccgccctt tagtgctgca gctaacgcat taagcactcc gcctggggag 840 tacggtcgca agagtgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 900 gtggtttaat tagaagcaac gcgaagaacc ttaccaggtc ttgacatcct ctgacaatcc 960 tagagatagg acgtcccctt cgggggcaga gtgacaggtg gagcatggtt gtcgtcagct 1020 cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatt ttagttgcca 1080 gcattcagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga 1140 cgtcaaatca tcatgcccct tatgacctgg gttacacacg tgttacaatg gacagaacaa 1200 agggcagcga aaccgcgagg ttaagccaat cccacaaatc tgttttcagt tcggatcgca 1260 gtctgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag catgccgcgg 1320 tgaatacgtt cccgggcctt gtacacaccg cccgtctctc cacgagagtt tgtaacaccc 1380 gaagtcggtg aggtaacctt ttaggagcca gccgccga 1418

Claims (7)

Bacillus amyloliquefaciens CJ 14-6 (KCCM 11718P) having excellent protease activity and anti-obesity activity separated and isolated from traditional meju. A step of growing soybeans by immersion or sowing on soybeans; And
The emulsifying step of inoculating and fermenting the thus-grown soybean with the Bacillus amyloliquefaciens strain CJ 14-6 of claim 1
&Lt; / RTI &gt; 3. The method of claim 2,
Further comprising the step of immersing the selected soybeans at 10 to 50 &lt; 0 &gt; C for 1 to 15 hours before the growing step,
The boiling step includes the step of adding saturated steam of 1.0 to 2.0 kgf / cm ² at 100 ° C to 150 ° C for 1 to 60 minutes,
Further comprising a step of cooling the soybeans to 30 to 50 &lt; 0 &gt; C after the increasing step. 3. The method of claim 2,
Wherein the growing step is carried out in a high pressure steam sterilizer (Autoclave) at 100 ° C to 150 ° C for 5 to 15 minutes. 3. The method of claim 2,
Wherein the Bacillus amyloliquefaciens CJ 14-6 strain is inoculated to the beans so that the amount of the added beans is 0.1 to 3.0 wt% based on the total amount of the raw materials. A soybean curd prepared by the method according to any one of claims 2 to 5. A soybean curd having an anti-obesity activity produced by the method according to any one of claims 2 to 5.